It is hypothesized that both intrinsic cellular properties and extrinsic microenvironmental conditions in human tumors play significant direct and indirect roles in determining basic tumor cellular characteristics which influence sensitivity to therapeutic modalities such as ionizing radiation. Little is known about these heterogeneous intrinsic and extrinsic properties of human tumor cells with respect to radiation sensitivity because, for the most part, such studies await the development of appropriate experimental models and techniques. Methods and experimental models need to be developed to characterize this heterogeneity, to evaluate mechanisms affecting biological behavior and responsiveness to therapy, to develop and evaluate new therapeutic strategies, and to predict for therapeutic responsiveness. The research program proposed here includes experiments designed to compare the usefulness and relevance for radiation therapy of multicell spheroids, clonogenic stem cell assays, and xenografts. The specific aims of the experiments are (1) to optimize and characterize the growth in these 3 model systems of cell isolates from selected human cancer (mainly squamous cell carcinoma), (2) to directly determine the influence of certain specific external environmental conditions on intra-spheroid oxygenation and on cellular heterogeneities, and (3) to use these in vitro and in vivo model systems to determine the importance to the overall radiation response of human tumors at different stages of growth of intrinsic cell sensitivity, intercellular contact, repair, differentiation, hypoxia, and altered growth kinetics such as the development of quiescent cell populations. The central general questions we are asking are (a) What are the relative strengths and limitations of the 3 model systems for growing and characterizing human cancer cells? (b) Do human tumor cells under experimental conditions in vitro reflect the important in vivo properties? (c) What assays and endpoints are most informative for characterizing clinically relevant biological behavior and for predicting therapeutic responsiveness? (d) What are some of the inter-patient tumor differences responsible for sensitive and resistant disease? (e) What therapeutic strategies using radiation alone or combined with drugs would be effective for resistant tumors? The research proposed represents the initial phases of a concentrated, collaborative effort to more directly study human cancer in appropriate model systems which can evaluate the significance of the dynamic nature of cellular and environmental diversity that occurs at different stages of tumor growth.